1. Field of the Invention
This invention relates to the construction and expression of gene fusions encoding a functional spinach acyl carrier protein-I (ACP-I) and the F.sub.c -binding portion of staphylococcal protein A. Additionally this invention relates to methods for the purification of enzymes participating in fatty acid synthetase (FAS) reactions.
2. Description of the Prior Art
Heretofor there has been difficulty in isolating and purifying FAS enzymes, particularly malonyl-CoA: Acyl Carrier Protein (ACP) transacylase (MCT) [Guerra, et al., Arch. Biochem. Biophys., Vol. 246, (1986), pp. 274-285].
Gene fusions encoding hybrid eukaryotic to prokoryotic proteins have been shown to stabilize as well as protect these proteins from proteolysis during expression in Escherichia coli (E. coli) [Davis, et al., Proc. Natl. Acad. Sci. USA, Vol. 78, (1981), pp. 5376-5380; Goeddel, et al., Proc. Natl. Acad. Sci. USA, Vol. 76, (1979), pp. 106-110 and Itakura, et al., Science, Vol. 49, (1984), pp. 483-492]. Fusions of this type can result in a higher level of eukaryotic protein production. Furthermore fusion to proteins such as beta-galactosidase or staphylococcal protein A allows for the affinity purification of the fusion protein [Geronimo, et al., Proc. Natl. Acad. Sci., USA, Vol. 80, (1983), pp. 6848-6852 and Nilsson, et al., EMBO J, Vol. 4, (1985), pp. 1075-1080]. The fusion of small proteins and peptides to larger proteins using recombinant DNA techniques serves ideally as an alternative to chemical coupling.
In plants ACP exists as a small acidic cofactor protein which participates in at least 12 reactions of fatty acid biosynthesis and metabolism and occurs in at least 2 isoforms (ACP I and ACP II) in several plants [Hoj PB, S. I., Carlsberg Res. Commun., Vol. 49 (1984), pp. 483-492; Ohlrogge, et al., The Biochemistry of Plants, Stumpf, P. K. (ed), Vol. 9, pp. 137-157, Academic Press, Orlando, Fla., (1987); Ohlrogge, et al., J. Biol. Chem., Vol. 260, (1985), pp. 8032-8037 and Ullman, A., Gene, Vol. 29, (1984), pp. 27-31]. In recent years, research on this protein has intensified because of the potential of ACP to serve as a representative marker protein for studies of the regulation of plant fatty acid synthetase gene expression. The cloned gene encoding spinach ACP-I has been sequenced [Beremand, et al., Arch. Biochem. Biophys., Vol. 256, (1987), pp. 90-100] and the subject of the current investigations.
In light of the prior art it can be seen there is a need for efficient methods by which proteins participating in fatty acid biothsynthesis may be identified and purified.